Recent studies describing the complicated cutting and rejoining reactions which occur during maturation of eukaryotic RNA emphasize the importance of specific ribonuclease and RNA in gene expression. A detailed analysis of the enzymes responsible will be necessary to determine their relative role in regulation of cellular processes. No such study of the RNA related enzymes of an mammalian cell has yet been published. We, therefore, propose to carry out a systematic survey of HeLa cell enzymatic activities which create or destroy covalent bonds in the phosphodiester backbone of RNA. Two striking observations made recently in our laboratory suggest to us that the proposed cataloguing of ribonuclease activities is not as formidable a task as it may, at first, appear: (a) HeLa cells grown and fractionated using conventional methods are remarkably free of random RNase activities; (b) an endonucleolytic activity capable of limited cleavage of hnRNA, but incapable of cleaving other large RNA test substrates such as the 800-base phage t7 early mRNA precursor, was detected in subcellular fractions. In carrying out the proposed study, we will (1) prepare specific RNA substrates (including synthetic polymers, unfractionated tRNA, rRNA and mRNA precursors, and highly purified precursors of individual RNAs) as probes for a wide array of RNA cleaving activities in HeLa cells; (2) establish rapid assay systems for detection of cleavage of these substrates; (3) use these substrates and their assays to identify any enzymatic activities which can be detected in crude extracts of HeLa cells; (4) carry out extensive subcellular fractionation of HeLa cells to determine the distribution and richest source of each activity and (5) develop purification schemes for selected enzymes.